Extrait du Journal de Chimie Physique, 1968, 65 no 1, p. 44.
ARE THERE PATHWAYS FOR PROTEIN FOLDING ?
by CYRUS LEVINTHAL
[Massachusetts Institute of Technology, Department of Biology Cambridge, Massachusetts.]
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SUMMARY
Denatured proteins, which have had essentially all
of their native three-dimensional structure disrupted,
can refold from their random disordered state into a
well-defined unique structure, in which the biological
activity is virtually completely restored. This experi-
mental result has lead to the suggestion that a native
protein exists in some kind of thermodynamic con-
figurational equilibrium, with the biologically active
state being the one with the lowest configurational
energy. An alternative view is that the native protein
is in a uniquely selected metastable state, in which
the configurational energy is at a local minimum. In
this latter model, the protein is not assumed to be in
the equilibrium state, and one must postulate some
sequence of events which takes place for each mole-
cule so that the protein reaches the correct metastable
state.
One possible sequential process which might lead
a protein to land in a particular state, is the growth of
the peptide chain on the ribosome, starting with the
amino terminal end and proceeding to the carboxy
terminus. Although one could imagine a protein
folding as it grows, and thus attaining a particular
metastable state, this is clearly not a necessary condi-
tion for correct folding, at least for those proteins
which have been shown to be reversibly denaturable.
However, the fact that folding on the ribosomes is not
necessary for the establishment of structure, does not
imply that any theory invoking a pathway of folding
can be eliminated. Such a pathway only requires
some local interactions or condensations of segments
of the polypeptide chain whenever the denatured
protein is put into the appropriate renaturing medium.
These segments would form unique three-
dimensional structures which make further conden-
sation more likely. Thus, a pathway of folding means
that there exist a well-defined sequence of events
which follow one another so as to carry the protein
from the unfolded random coil to a uniquely folded
metastable state. If the final folded state turned out to
be one of lowest configurational energy, it would be
a consequence of biological evolution not of physical
chemistry.
Three approaches have been used in investigating
this problem. First, the refolding and dimerization of
the enzyme alkaline phosphatase obtained from the
bacterium E. Coli has been studied under varying
conditions and from a variety of mutant strains. Mu-
tants have been selected which fail to make active
enzyme at 44C. About half of these mutants have
activity when the cells are grown at 25C, and the
enzyme produced at the low temperature has been
found to be stable even at low temperatures much
higher than that used in the selection. Thus, these
mutants have a temperature-sensitive step in one of
the events which normally leads to the formation of
active enzyme, but the enzyme produced is not tem-
perature sensitive.
A second approach involved the use of computer-
aided molecular model building in attempts to deduce
plausible pathways which proteins can follow as they
are folding. Starting with an amino acid sequence we
can describe the configuration of the protein i.e., the
position of each of its atoms in space if we know the
dihedral angles for the backbone and, in addition, the
rotation angle about the appropriate bonds of the
amino acid residues. Using a computer controlled
display system, the molecule thus generated can be
displayed in such a way that the observer can see the
three-dimensional relationships in the structure.
Computer programs have been written in such a way
that any configuration can be altered to minimize the
Van der WAALS energy and to insure close packing
of the structure. However, this energy minimization
can only be expected to alter the structure to the bot-
tom of the local minimum; it is not intended to search
through all possible configurations for a true mini-
mum energy. In addition, the investigator can alter
the computer generated structure as if he were deal-
ing with physical models in which one part could be
pushed or pulled relative to another. Thus, the com-
puter-aided model building is not designed to find the
configuration of minimum energy rather, it is de-
signed as an aid to the investigator as various se-
quentially folding steps are tried.
__________________
This system has been used in an attempt to obtain
such a pathway of folding for the protein cytochrome
C. A plausible structure has been obtained in this way
which satisfies all of the known chemical interactions
of the molecule. However, the uniqueness of the pro-
posed folding process has not been determined.
Finally, the computer system has been used in at-
tempts to deduce plausible folding pathways for
myoglobin and lysozyme. Three-dimensional pictures
of the structures and some of the folding sequences
will be shown.
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